Dyballa Nadine, Metzger Sabine
Biological-Medical-Research Center, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany.
Methods Mol Biol. 2012;893:47-59. doi: 10.1007/978-1-61779-885-6_4.
Proteins separated by two-dimensional gel electrophoresis can be visualized by in-gel detection using -different staining methods. Ideally, the dye should bind non-covalently to the protein following a linear response curve. Since protein concentrations in biological systems may vary by six or more orders of magnitude (Corthals GL et al., Electrophoresis 21(6):1104-1115, 2000), the staining should allow for a detection of very low protein amounts. At the same time, saturation effects have to be avoided because they impede normalized quantification.Most proteomics laboratories apply Coomassie, silver, or fluorescent stains. Using the colloidal properties of Coomassie dyes, detection limits at the lower nanogram level can meanwhile be achieved. Characteristics like ease of use, low cost, and compatibility with downstream characterization methods such as mass spectrometry, therefore, make colloidal Coomassie staining well suited for the in-gel detection method in quantitative proteomics.
通过二维凝胶电泳分离的蛋白质可以使用不同的染色方法通过凝胶内检测进行可视化。理想情况下，染料应按照线性响应曲线与蛋白质非共价结合。由于生物系统中的蛋白质浓度可能相差六个或更多数量级（Corthals GL等人，《电泳》21(6):1104 - 1115，2000），染色应能够检测到非常低的蛋白质量。同时，必须避免饱和效应，因为它们会妨碍归一化定量。大多数蛋白质组学实验室使用考马斯亮蓝、银染或荧光染色。利用考马斯亮蓝染料的胶体性质，同时可以实现低纳克水平的检测限。因此，诸如易用性、低成本以及与质谱等下游表征方法的兼容性等特性，使得胶体考马斯亮蓝染色非常适合用于定量蛋白质组学中的凝胶内检测方法。
